The long-term objective of this project is to understand the enzymatic properties of the vitamin K epoxide reductase (VKOR) and its role in vitamin K-dependent carboxylation. Carboxylation is an essential post-translational modification for proteins important in several physiologic functions, including blood coagulation and bone metabolism. Recently, researchers in this laboratory identified the warfarin-sensitive VKOR gene. VKOR is the target of warfarin, the most widely prescribed anti-coagulant for thromboembolic disorders. Although estimated to prevent twenty strokes per induced bleeding episode, warfarin is under-utilized because of fear of bleeding. Research funded by this grant hopefully will lead to therapies that will alleviate some of the problems involved in warfarin treatment. Specifically, we propose to accomplish the following during the tenure of this grant: (1) express the isoforms of the VKOR gene to determine which have activity;(2) purify the VKOR using the amino acid tag we have inserted and the antibody to this tag;(3) investigate different cell expression systems to determine which is best for production of recombinant VKOR;(4) investigate the status of cysteines in VKOR, the glycosylation sites and other potential post-translational modifications, and the warfarin binding site by mass spectrometry;and (5) investigate the effect of vitamin K epoxide reductase (VKOR) gene polymorphisms on response to warfarin. This study of VKOR should lead to a better understanding of the mechanism of warfarin inhibition. It may also result in more accurate dosing of warfarin and in the design of new anti-coagulants.